In an effort to probe whether the metal content of metallo-β-lactamase L1 is affected by metal ion bioavailability, L1 was over-expressed as mature protein (M-L1) and full-length (FL-L1) analogs, and the analogs were characterized with metal analyses, kinetics, and EPR spectroscopy. FL-L1, containing the putative leader sequence, was localized in the periplasm of E. coli and shown to bind Zn(II) preferentially. The metal content of FL-L1 could be altered if the enzyme was over-expressed in minimal medium containing Fe and Mn, and surprisingly, an Fe-binding analog was obtained. On the other hand, M-L1, lacking the putative leader sequence, was localized in the cytoplasm of E. coli and shown to bind various amounts of Fe and Zn(II), and like FL-L1, the metal content of the resulting enzyme could be affected by the amount of metal ions in the growth medium. L1 was refolded in the presence of Fe, and a dinuclear Fe-containing analog of L1 was obtained, although this analog is catalytically-inactive. EPR spectra demonstrate the presence of an antiferromagnetically-coupled Fe(III)Fe(II) center in Fe-containing L1 and suggests the presence of a Fe(III)Zn(II) center in M-L1. Metal analyses on the cytoplasmic and periplasmic fractions of E. coli showed that the concentration of metal ions in the periplasm is not tightly controlled and increases as the concentration of metal ions in the growth medium increases. In contrast, the concentration of Zn(II) in the cytoplasm is tightly-controlled while that of Fe is less so.Bacterial resistance to β-lactam containing antibiotics such as penicillins, cephalosporins, and carbapenems is most often accomplished by expression of β-lactamases, which hydrolyze the C-N bond of these antibiotics ( 1-4 ). A majority of these β-lactamases utilize an active site serine group for the nucleophilic attack on the β-lactam carbonyl, and the serine β-lactamases have been studied extensively for many years ( 4 ). On the other hand, one class (Class B) of β-lactamases utilizes a metal-assisted hydrolysis pathway to inactivate β-lactam containing antibiotics, and these enzymes are called metallo-β-lactamases (mβl's) ( 1, 2, 5-7 ). The mβl's have been further divided into subgroups based on sequence identity, Zn(II) content, substrate preference, and biochemical properties. Subgroup B1 enzymes require 2 Zn(II) ions for full catalytic activity, exhibit kinetic preference for penicillins as substrates, exhibit >23% sequence identity toward other subgroup B1 members, and are represented by mβl's CcrA from Bacteroides fragilis, BcII from Bacillus cereus, and IMP-1 from various sources ( 1, 5 ). Subgroup B2 enzymes require only 1 Zn(II) ion for full catalytic activity, preferentially hydrolyze carbapenems, exhibit 11% sequence identity with the subgroup B1 enzymes, and